D-alanine ligase (Yersinia pestis)

Target: D-alanine D-alanine ligase
NCBI Gene # or RefSeq#: NC_003143.1
Protein ID or Wolbachia#: YP_002345628.1
Organism: Yersinia pestis
Etiologic Risk Group: Risk Group 3
Disease Information: Yersinia pestis is a bacterium that causes a variation of plague in humans and other mammals. Humans typically get plague after being bitten by a rodent flea that is carrying the plague bacterium, encountering contaminated fluids or tissue, or inhaling infections droplets. Plague is notorious for killing millions of people in Europe during the Middle Ages. Since that time, plague has occurred in rural and semi-rural areas of the western United States. Without immediate treatment, the disease can cause serious illness or death. The most common form of plague is bubonic plague, which infects the lymphatic system and causes inflammation. When the bacteria enter the bloodstream, the disease is then recognized as septicemic plague. Alternatively, if the bacteria spread to the lungs, the disease is then considered to be pneumonic plague, which is the only form of plague that can be transmitted between people. One of the enzymes that helps Yersinia pestis infect its host is D-alanine D-alanine ligase, which synthesizes D-alanyl-D-alanine, an essential precursor of bacterial peptidoglycan. For this reason, D-alanine ligase is an important target for the development of antibacterial drugs.
Link to TDR Targets page: N/A
Link to Gene Database page: https://www.ncbi.nlm.nih.gov/gene/1173401
Essentiality of this Protein: D-alanine D-alanine ligase is an essential enzyme in bacterial cell wall biosynthesis.
Is it a monomer or multimer as biological unit? Multimer
Complex of Proteins? D-alanine D-alanine ligase consists of three domains, in which four loops, loop 1, loop 2, loop 3 and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule.
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): https://www.ncbi.nlm.nih.gov/pubmed/23286234
EC#: 6.3.2.4
Link to BRENDA EC# page: https://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.4&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Yersinia+pestis&show_tm=0

Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): http://vdsstream.wikispaces.com/file/view/KovacDalanineDalanineLigaseVirtualScreenJMedChem2008.pdf/261437724/KovacDalanineDalanineLigaseVirtualScreenJMedChem2008.pdf
Link to Sigma page for Assay: http://scripts.iucr.org/cgi-bin/paper?S2059798315021671
http://www.sigmaaldrich.com/catalog/product/sigma/a7377?lang=en®ion=US
Substrate Cost: $37.40
Substrate Quantity: 5G
Substrate Catalog #: A7377-5G
PDB Structure: 5C1P

Current Inhibitors:
http://vdsstream.wikispaces.com/file/view/KovacDalanineDalanineLigaseVirtualScreenJMedChem2008.pdf/261437724/KovacDalanineDalanineLigaseVirtualScreenJMedChem2008.pdf
Expression Information (has it been expressed in bacterial cells): D-alanine D-alanine ligase has been expressed in E. coli.
Purification Method: Ni-NTA
Image of Protein:

D-alanine D-alanine Ligase.png

Amino Acid Sequence:

MAEKVAVLLGGTSAEREVSLLSGQAVLAGLKEAGIDAYGVDTKDFPVTQLKEQGFDKVFI
ALHGRGGEDGTLQGVLEFLQLPYTGSGVMASALTMDKLRTKLVWQALGLPISPYVALNRQ
QFETLSPEELVACVAKLGLPLIVKPSHEGSSVGMSKVDHASELQKALVEAFQHDSDVLIE
KWLSGPEFTVAILGDEVLPSIRIQPPGVFYDYDAKYLSDKTQYFCPSGLSDESEQQLAAL
ALQAYHALDCSGWGRVDVMQDRDGHFYLLEVNTSPGMTSHSLVPMAARQYGLSFSQLVAR
ILMLADX

Length of your protein in Amino Acids: 307 AA
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 33261.23
Molar Extinction coefficient of your protein at 280 nm wavelength: 31525
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html):

CDS Gene Sequence:

<span style="font-family: monospace,serif;">>NC_003143.1:602769-603689 Yersinia pestis CO92 chromosome, complete genome
<span class="ff_line">ATGGCTGAGAAAGTCGCGGTATTGCTGGGTGGGACCTCTGCGGAGCGTGAAGTGTCATTACTGTCAGGTC
AGGCGGTATTGGCAGGCTTGAAAGAAGCCGGTATCGATGCTTATGGGGTGGATACCAAAGATTTTCCGGT
AACTCAGCTTAAAGAACAGGGCTTTGATAAAGTGTTTATCGCCCTGCATGGTCGTGGTGGCGAAGACGGT
ACGTTACAAGGCGTATTGGAATTTCTACAACTGCCATATACCGGCAGTGGTGTAATGGCATCCGCCCTGA
CAATGGACAAATTGCGCACCAAATTAGTCTGGCAAGCCTTGGGGTTGCCTATTTCGCCTTATGTTGCGTT
GAATCGCCAACAGTTTGAAACACTTTCCCCAGAGGAATTGGTGGCGTGTGTCGCTAAGCTTGGCCTGCCA
CTTATTGTCAAGCCAAGCCATGAAGGCTCTAGCGTTGGGATGAGTAAAGTTGATCACGCCAGCGAATTGC
AGAAGGCATTGGTTGAAGCTTTCCAACATGACAGCGATGTACTGATTGAGAAATGGCTGAGCGGGCCAGA
GTTTACTGTCGCGATCCTTGGGGATGAAGTTTTGCCTTCTATTCGTATTCAGCCTCCGGGCGTGTTTTAC
GATTATGACGCTAAGTATTTGTCAGATAAAACACAATACTTCTGCCCAAGCGGTTTAAGTGATGAGTCGG
AGCAACAATTGGCAGCGCTGGCATTGCAGGCTTACCACGCGCTGGATTGCAGCGGTTGGGGGCGGGTCGA
TGTGATGCAAGACCGCGATGGTCATTTTTACCTGCTTGAAGTGAACACTTCACCGGGTATGACCAGCCAT
AGTTTGGTTCCAATGGCCGCCCGCCAGTATGGGTTGAGTTTCTCCCAATTGGTGGCGCGCATTCTGATGT
TGGCTGACTGA</span></span>

GC% Content for Gene: N/A
CDS Gene Sequence (codon optimized):

<span style="background-color: #ffffff; font-family: 'Courier New',Courier,monospace,arial,sans-serif; font-size: 14px;">  1 ATGGCGGAAAAAGTTGCGGTTCTC  24
  2 ACAGCAGAGAAACTTCACGTTCCGCAGAGGTACCACCCAGGAGAACCGCAACTTTTTCCG  60
  3 AACGTGAAGTTTCTCTGCTGTCTGGTCAGGCAGTTCTGGCGGGTCTGAAAGAAGCTGGCA  60
  4 GGTAACCGGGAAGTCTTTGGTGTCAACACCGTACGCGTCGATGCCAGCTTCTTTCAGACC  60
  5 CCAAAGACTTCCCGGTTACCCAGCTGAAGGAACAGGGTTTCGACAAAGTTTTCATCGCTC  60
  6 CGCCCTGGAGGGTACCGTCTTCACCACCACGACCGTGCAGAGCGATGAAAACTTTGTCGA  60
  7 GGTACCCTCCAGGGCGTTCTGGAGTTCCTGCAGCTCCCGTACACCGGTTCTGGTGTTATG  60
  8 CAGTTTGGTGCGCAGTTTGTCCATGGTCAGTGCGCTCGCCATAACACCAGAACCGGTGTA  60
  9 CAAACTGCGCACCAAACTGGTTTGGCAGGCGCTGGGCCTGCCGATCTCTCCATACGTTGC  60
 10 TTCTTCCGGAGACAGCGTTTCGAACTGCTGACGGTTCAGCGCAACGTATGGAGAGATCGG  60
 11 ACGCTGTCTCCGGAAGAACTGGTAGCGTGCGTTGCGAAACTGGGTCTGCCGCTGATCGTG  60
 12 CAACTTTAGACATACCAACAGAAGAACCTTCGTGAGACGGTTTCACGATCAGCGGCAGAC  60
 13 TCTTCTGTTGGTATGTCTAAAGTTGACCACGCCTCTGAACTGCAAAAAGCGCTGGTTGAA  60
 14 AGCCATTTTTCGATCAGAACGTCAGAGTCGTGCTGGAACGCTTCAACCAGCGCTTTTTGC  60
 15 CGTTCTGATCGAAAAATGGCTGAGCGGTCCGGAATTCACCGTTGCTATCCTGGGCGACGA  60
 16 GTAGAAAACACCTGGCGGCTGGATACGGATAGACGGCAGAACTTCGTCGCCCAGGATAGC  60
 17 GCCGCCAGGTGTTTTCTACGACTACGACGCGAAATACCTGTCTGACAAGACGCAATACTT  60
 18 GCTGCTGTTCAGATTCATCGGACAGGCCAGACGGGCAGAAGTATTGCGTCTTGTCAGACA  60
 19 CGATGAATCTGAACAGCAGCTCGCGGCACTGGCGCTGCAAGCGTACCACGCGCTGGACTG  60
 20 ACCGTCACGGTCCTGCATAACGTCAACGCGACCCCAACCAGAGCAGTCCAGCGCGTGGTA  60
 21 GCAGGACCGTGACGGTCACTTCTACCTGCTCGAAGTTAACACGAGCCCGGGTATGACCTC  60
 22 AGACAGACCGTACTGACGTGCTGCCATCGGTACCAGAGAGTGAGAGGTCATACCCGGGCT  60
 23 ACGTCAGTACGGTCTGTCTTTCTCTCAGCTCGTAGCGCGTATCCTGATGCTGGCGGACTA  60
 24 TTAGTCCGCCAGCATCAGG  19</span>

GC% Content for Gene (codon optimized): N/A
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
DNA Works File: https://drive.google.com/drive/folders/0B4O2KqKh2q_-dFJuSVJtcDgtUUU
Primer Design Results for 'Tail' Primers:
YpDAL_for: TACTTCCAATCCATGGCGGAAAAAGTTGCGGTTCT
YpDAL_rev: TATCCACCTTTACTGTTAGTCCGCCAGCATCAGGA